The objective of this work was to study the ability of blood cells and their microparticles to transport monomeric and pentameric forms of C-reactive protein (mCRP and pCRP) in the blood of patients with coronary artery disease (CAD). Blood was obtained from 14 patients with CAD 46 ± 13 years old and 8 healthy volunteers 49 ± 13.6 years old. Blood cells and microparticles with mCRP and pCRP on their surface were detected by flow cytometry. Messenger RNA (mRNA) of CRP was extracted from peripheral blood monocytes stimulated with lipopolysaccharide (LPS) and granulocyte-macrophage colony-stimulating factor (GM-CSF). mRNA of CRP in monocytes was detected with PCR. Monocytes were predominantly pCRP-positive (92.9 ± 6.8%). mCRP was present on 22.0 ± 9.6% of monocyte-derived exosomes. mCRP-positive leukocyte-derived microparticle counts were significantly higher (8764 ± 2876/µL) in the blood of patients with CAD than in healthy volunteers (1472 ± 307/µL). LPS and GM-CSF stimulated monocytes expressed CRP mRNA transcripts levels (0.79 ± 0.73-fold), slightly lower relative to unstimulated hepatocytes of the HepG2 cell line (1.0 ± 0.6-fold), but still detectable. The ability of monocytes to transport pCRP in blood flow, and monocyte-derived exosomes to transmit mCRP, may contribute to the maintenance of chronic inflammation in CAD. Monomeric C-reactive protein (mCRP) is a form of C-reactive protein (CRP), which may possess pro-inflammatory properties . It is fWhether mCRP is synthesized locally in areas of inflammation or transported therein from blood flow, remains a subject of discussion. Several papers argue in favor of local CRP synthesis by macrophages. Kaplan et al. showed that monocyte-derived macrophages express CRP mRNA . Dong etThe evidence that mCRP may be present in circulating blood is sparse. To date, it is known that mCRP can be transported in blood flow on microparticles ,18. Othevena cubitalis in S-MonovetteTM vials  with 3.2% sodium citrate at blood to anticoagulant ratio 1:9. Then, samples were transferred into separate tubes to perform the study in whole blood and platelet-poor plasma (PPP). PPP was prepared by centrifugation for 20 min at 2000 g. Samples were immediately prepared for flow cytometry as described further. Informed written consent was given by patients according to the Declaration of Helsinki, and the study was approved by the Ethics Committee of the National Medical Research Centre of Cardiology .Blood samples were collected from 14 male and female patients with CAD 46 ± 13 years old and 8 healthy volunteers 49 ± 13.6 years old. The clinical characteristic of patients, and inclusion and exclusion criteria are provided in To distinguish between mCRP and pCRP, we combined the FITC-labeled monoclonal antibody to pCRP (mAb to pCRP), clone 372 , and PE-labeled mAb to mCRP with cross-reactivity to pCRP (mAb to mCRP), clone 328 . The description of the production of these mAbs is provided in the TM exosome capture kit and detected with EXOFITCTM exosome stain  according to the manufacturer’s guide. As both the exosome stain and mAb to CD63 were FITC-labeled, we performed staining of each sample in separate tubes. We used a non-specific FITC-labeled mouse IgG as a negative control. In each test at least 10,000 events were registered.To detect different types of blood cells and their microparticles, we combined PE-Cy7-labeled mAb to CD235a, PerCP-Cy5.5-labeled mAb to CD45, APC-labeled mAb to CD41, APC-R700-labeled mAb to CD14, FITC-labeled mAb to CD63 , V450-conjugated Annexin-V, and 1 µm latex beads  as a size reference. Exosomes were isolated with the Exo-FLOWTM II flow cytometer  on a day of sample collection. The threshold level of 200 units was chosen for Side Scatter to exclude debris and artifacts. Analysis of flow cytometry results was done on FACSDivaTM software . The same antibody panel was used to detect cells and microparticles. To calculate the number of microparticles per µL, we used BD TrucountTM  tubes as a reference.Detection of cells, microparticles, and exosomes in samples were performed on the FACSCantoMonocytes were isolated and cultured according to a standard protocol. The detailed description is provided in the The cell line of hepatocellular carcinoma of human HepG2 was kindly provided by P.N. Rutkevich . The detailed description is provided in the TM Mini Kit  according to the manufacturer’s protocol. RNA concentrations were measured with a spectrophotometer Nanodrop  and quality was assessed by agarose gel electrophoresis. The obtained RNA in an amount of 3 μg was used to synthesize coding DNA (cDNA) with oligo(dT) primer using the MaximaTM First Strand cDNA Synthesis Kit  according to the manufacturer’s protocol. PCR was performed with the SYBRTM Green intercalating dye  in a Step One Plus™ Real-Time PCR System amplifier . As primers, unique pairs of oligodeoxynucleotides complementary to the analyzed mRNA/cDNA were used . The Mann–Whitney U test was used to compare two groups of data and the Kruskal–Wallis ANOVA by ranks test to compare three or more samples. Differences were regarded as statistically significant if the null hypothesis was rejected with a probability >95%. The data were analyzed on the STATISTICA software v.6.0 .We used two antibodies to detect CRP: FITC-labeled mAb with verified selective reactivity to pCRP and PE-labeled mAb to mCRP with cross-reactivity to pCRP. The flow cytometry results, in which the signal from mAb to pCRP (FITC+/PE–) or both mAbs to pCRP and mCRP (FITC+/PE+) was present, were considered pCRP-positive. The flow cytometry results, in which the signal from mAb to pCRP was absent and the signal from mAb to mCRP was present (FITC–/PE+), were considered mCRP-positive . We studThen, we detected CRP on the surface of blood cells. As a negative control, we used the non-specific FITC-labeled mouse IgG. p < 0.05, Kruskal–Wallis ANOVA by ranks test).All obtained results were FITC+/PE+, meaning that pCRP, but not mCRP, was present on the surface of blood cells. As shown in We identified the subtype of pCRP-positive leukocytes. On flow cytometry diagrams, acquired after erythrocyte lysis, we selected CD45-positive leukocytes a. From tTM exosome capture kit and mAb to exosome-specific cytoskeletal protein CD63. As seen in Next, we studied blood cell-derived microparticles in PPP. Following the established approach, we assumed that microvesicles are subcellular particles produced through outward blebbing of the plasma membrane of cells, and predominantly bear plasma membrane proteins; exosomes are particles produced in intracellular space, capturing cytoplasm content, including proteins, RNAs, and other intracellular entities . We applp < 0.05, Kruskal–Wallis ANOVA by ranks test). We isolated leukocyte microparticles with Exo-FLOWTM exosome capture kit and then stained with exosome-specific stain ExoFITCTM. There was a distinct group of ExoFITCTM-positive, mCRP-positive exosomes (22.0 ± 9.6%). To double-check our result, we stained PPP samples with mAb to exosome-specific cytoskeletal protein CD63: 20.8 ± 11.2% of microparticles were CD63-positive, mCRP-positive.According to Annexin-V staining, most erythrocyte and a substantial part of platelet microparticles were Annexin-V positive microvesicles: 72.7 ± 11.9% of erythrocyte microparticles and 27.8 ± 6.4% of platelet microparticles, but only 3.5 ± 1.6 of leukocyte microparticles were annexin-V-positive (n = 14) than in the blood of healthy volunteers . The difference between the results was statistically significant . This study did not investigate the relationship between the severity of CAD and CRP-positive cells or microparticle numbers.We measured mCRP-positive CD45-positive microparticle counts in the blood of 14 patients with CAD and 8 healthy volunteers. In the blood of patients with CAD, mCRP-positive CD45-positive microparticle counts were higher . mRNA levels of CRP between probes were normalized by the mRNA levels of β-actin as a housekeeping gene. The expression of CRP was quantified with RT-PCR in LPS, GM-CSF stimulated monocytes, IL-6-stimulated and unstimulated hepatocytes of the HepG2 cell line. The data presented as the relation of the expression of CRP mRNA in stimulated monocytes and stimulated HepG2 hepatocytes to the expression in unstimulated HepG2 hepatocytes. As shown in In addition, CRP mRNA expression was validated by electrophoresis of amplicons in 1.5% agarose gel. The sizes of the PCR products that corresponded to CRP mRNA were 167 and 245 base pairs. The PCR product for the housekeeping gene ActB was 144 bp. Both CRP transcripts (245 base pairs and 167 base pairs) were detected at low levels in stimulated monocytes . CRP traThere is growing evidence that mCRP can be detected in circulating blood. Habersberger et al. identified mCRP on microparticles circulating in the blood of persons with acute myocardial infarction (AMI) . Later, Taking into consideration the evidence that mCRP can be detected in circulating blood, particularly on microparticles, we studied blood cells and microparticles to identify which of them bear mCRP. Monocytes had the largest population of CRP-bearing cells. These cells presented a pentameric, but not monomeric form of CRP. The presence of pCRP on monocyte membranes may facilitate the process of dissociation that occurs in vivo ,12. In tWe also detected pCRP on a substantial number of circulating cell-derived microparticles, mainly on Annexin-V positive microvesicles, and mCRP on annexin-V negative leukocyte-derived exosomes. Microvesicles are fragments of cell membranes, shed upon cell activation. Microvesicles are much smaller than cells, with size usually varying from 0.1 to 1 µm in diameter. In the circulation, they bear signal molecules and have antigen-presenting capabilities. They are involved in immunomodulation as either pro-inflammatory or anti-inflammatory agents ,23. MicrExosomes belong to another group of cell-derived microparticles. Exosomes are usually smaller than microvesicles, with a size of less than 0.2 µm. Unlike microvesicles, which originate from cell membranes, exosomes are produced in the endocytic-lysosomal system of cells and released by exocytosis. Exosome cargo can contain proteins, RNA, and other molecules of intracellular origin. In the blood flow, exosomes may act as transporters for this potentially regulatory cargo to target cells . AccumulThe emerging data show that mononuclear cells are able to transport other regulating proteins in CAD as well, such as the adenosine A2A receptor . This siPreviously, we reported higher counts of mCRP-positive leukocyte-derived microparticles in the blood of patients with CAD, compared to healthy volunteers . In thisWe studied the expression of CRP mRNA in LPS and GM-CSF-stimulated monocytes isolated from human peripheral blood. The level of expression was slightly lower relative to HepG2 but still detectable. We hypothesize that in vivo recruited monocytes/macrophages, stimulated in areas of local inflammation, may synthesize CRP directly in monomeric form, or in pentameric form, which may then undergo dissociation. Being presented on the membrane surface or shed with microparticles, it may influence the inflammatory response. This hypothesis is in line with the existing data. We have already mentioned papers that describe the expression of CRP mRNA by monocyte-derived macrophages , alveolan = 14), than in the blood of healthy volunteers . CRP mRNA expression by monocytes, the presence of mCRP on monocyte-derived exosomes and higher counts of mCRP-positive leukocyte-derived microparticles in the blood of patients with CAD, compared to healthy volunteers, may indicate the involvement of mCRP in the maintenance of the inflammatory response.Our results show that pCRP does not exist in blood flow only in a soluble form, but intensively binds to blood cells, and appear on their microparticles. Among blood cells, leukocytes had the largest pCRP-positive population ; 92.9 ± 6.8% of monocytes were pCRP-positive. mCRP presence was less evident, except for a large group of monocyte-derived exosomes, which comprised up to 22.0 ± 9.6% of the whole population. We found that GM-CSF and LPS-stimulated monocytes expressed CRP transcripts at a low but detectable level, which means that they are able to express CRP mRNA. In the blood of patients with CAD, mCRP-positive CD45-positive microparticle counts were significantly higher (8764 ± 2876 particles per µL, The method used in this work for the measurement of monomeric CRP with a combination of selective mAb to pCRP and cross-reactive mAb to mCRP and pCRP was patented in 2019. Patent RU2704128C1 “Method for measuring concentration of monomer C-reactive protein on the surface of blood cells”.


Qr) in the tissues. (3) Results: Permeation of CP-NPs is more effective in terms of drug retention in almost all tissues . In vivo studies show that neither of the two formulations affects tissue structure, so both formulations are safe. (4) Conclusions: It was concluded that CP-NPs may be a useful tool for the topical treatment of local inflammation in veterinary and human medicine.(1) Background: Carprofen (CP), 2-(6-chlorocarbazole) propionic acid, is used as an anti-inflammatory, analgesic and anti-pyretic agent and it belongs to the family of non-steroidal anti-inflammatory drugs (NSAIDs). CP has some adverse reactions in systemic administration; for this reason, topical administration with CP nanoparticles (CP-NPs) can be an optimal alternative. The main objective of this work is the investigation of ex vivo permeation of CP through different types of porcine mucous membranes  and ophthalmic tissues  to compare the influence of CP-NPs formulation over a CP solution (CP-Solution). (2) Methods: The ex vivo permeation profiles were evaluated using Franz diffusion cells. Furthermore, in vivo studies were performed to verify that the formulations did not affect the cell structure and to establish the amount retained ( Non-steroidal anti-inflammatory drugs (NSAIDs) are some of the most commonly used medications in human and veterinary medicine. Carprofen (CP), 2-(6-chlorocarbazole) propionic acid, is used as an anti-inflammatory, analgesic and anti-pyretic agent ,2. CP waThe main effect of CP is the inhibition of cyclooxygenase (COX), an important enzyme in the arachidonic acid cascade. This process generates other mediators involved in the inflammatory response, such as prostaglandins and thromboxanes . In veteNumerous localized pathologies in pigs could be treated topically such as ophthalmic diseases that occur with inflammation: entropion, distichiasis, trichiasis, lagophthalmia, foreign bodies, wounds, dry keratoconjunctivitis, infections, blepharitis and conjunctivitis ,25. ManyThe investigation of most of those pathologies in pigs is reasonably predictive of results in humans. Therefore, the study of anti-inflammatories in this species can provide much information for both veterinary and human medicine.The suitability of a drug to penetrate through the mucous membrane is correlated with its physicochemical properties, as well as, the pharmaceutical formulation ,30,31. OAnother aspect to consider when treating a localized topical inflammation concerns formulation effect. It must be ensured that the drug reaches the site of action at therapeutic concentrations and, in addition, these concentrations are maintained for a prolonged time to reduce inflammation. In this sense, the use of nanoparticles (NPs) can be a good alternative to conventional drugs. They represent promising drug carriers for topical applications . For topThe main objectives of this work are the investigation of ex vivo permeation of CP through different types of porcine mucous membranes or commonly involved ophthalmic tissues to compare the influence of a PLGA NP formulation ,36 over ®) of Merial-Boehringer Ingelheim ; Xylazine (Rompun®) of Bayer Hispania ; Midazolam Gen® (Midazolam); Propofol Lipuro 1%® (Propofol); Forane® (Isoflurane), Pentothal®  all of them from Centauro Veterinary . Endotracheal tubes with a low-pressure balloon, number 7 of Centauro Veterinary. All other chemicals were of analytical grade and used without further purification.CP was obtained from Capot Chemical  and NPs of CP were prepared as described before ,36. DeioPolymeric NPs of CP were transferred by the team of Parra et al. . Resultad,l-lactic-co-glycolic) acid 75:25  and purchased from Evonik Industries . Poloxamer 188 (Lutrol®F68) was from BASF . HP-CD was obtained from Sigma-Aldrich  and CP from Capot Chemical . Double distilled water was obtained from a MilliQ® Plus System, lab supplied. All other chemicals were of analytical grade and used without further purification.As explained in the study by Parra et al. , the matAs detailed in the study of Parra et al. , NPs of The team of Parra et al.  describeThree different types of porcine mucous were used as permeation membranes: buccal, sublingual and vagina. Additionally, three ophthalmic structures  were also used to evaluate CP permeation and/or penetration. Ex vivo tissues were obtained under veterinary supervision from residual individuals of female pigs , previously used in surgical university practices and according to the Ethics Committee of Animals Experimentation at the University of Barcelona. Pigs were anesthetized with intramuscular administration of ketamine (3 mg/kg), xylazine (2.5 mg/kg) and midazolam (0.17 mg/kg). After chirurgical experimentation, the animals were euthanized with an intravenous overdose of sodium thiopental. After debridation, samples were frozen to −20 °C and longitudinally cut in 700 µm slabs with a dermatome GA 630  ,39,40. T2 for all membranes except for buccal mucous (2.54 cm2). A dialysis membrane  was used according to Parra et al. [Ex vivo permeation study of NPs was performed in vertical Franz diffusion cells  following a previously validated procedure  with a da et al. .Mucous membrane samples were placed between the receptor and donor compartments with the proximal side in contact with the receptor medium and the mucous side in contact with the donor chamber. Samples of 300 µL were placed in the donor compartment (CP-Solution and CP nanoparticles (CP-NPs)). As receptor medium, PBS at pH 7.4 was used and kept at 32 +/− 0.5 °C under continuous stirring in accordance with sink conditions. Samples of 300 µL were withdrawn from the receptor compartment at pre-selected times for 6 h and replaced by an equivalent volume of fresh PBS at the same temperature. This experiment was done in six replicates for each tissue and formulation. At the end of the experiment, samples were analyzed by HPLC–UV.® software . A reverse-phase column C18, 3.9 × 150 nm packed with 5 µm, was used . The mobile phase, previously filtered by a 0.45 µm nylon membrane filter  and degassed by sonication, consisted of methanol and potassium dihydrogen phosphate diluted in 1 L distilled water PH = 3 (75:25). The injection volume was 30 µL and the flow rate was 0.1 mL/min. This method has been validated in our laboratory and is specific and sensitive for the detection of carprofen. The retention times were 2.4–3.6 min as we can see in The HPLC–UV apparatus consisted of a Waters LC Module I plus , with an ultraviolet detector set up at 235 nm, and MilleniumJs in mg/h cm2) across mucous membranes and the permeability coefficients (Kp in cm/h) were calculated at the steady state. Lag times (Tl) and Js values were calculated by linear regression analysis using the Graph Pad Prism® software version 5.01  [Qt is the amount of CP which permeates into the receptor compartment (mg). A is the active cross-sectional area available for diffusion (cm−2) and t is the time of exposure (h) per unit areaFor the permeation assays, the flux values per unit area  . StationKp, cm/h) was calculated based on the relationship:Js is the flux calculated per unit area at the steady state and oC is the drug concentration in the donor compartment.The permeability coefficient (P1) and diffusion parameter (P2) were estimated from the following equations:Partition parameter (Css) of drug that would penetrate mucous barrier after topical application, was obtained using the following equation:Css is the plasma steady-state concentration, Js the flux/area obtained in this study, A the hypothetical area of application and Clp the plasmatic clearance. Calculations were addressed on the basis of a maximum application area of 1 cm2 and Clp values of 2.18 L/h +/− 0.42.The predicted steady-state plasma concentration  was calculated with the following formula:Ex (mcg) is the amount of drug extracted, Px (g) is the weight of the permeated mucous membranes, A (cm2) is the active cross-sectional area available for diffusion and R is the percentage of recovery of the drug, obtained as described formerly [The retained amount of drug in the tissue  assessed the significance of the differences between formulations and groups of membranes. A p-value < 0.05 was accepted as significant.Nonparametric analysis was performed in these studies because drug permeation through animal tissues follows more closely a log-normal than a normal (Gaussian) distribution . NonparaIntegrity of the mucous tissues after in vivo formulations application was investigated with optical microscopy. The studies were conducted under a protocol approved by the Animal Experimentation Ethics Committee of the University of Barcelona (Spain) with ethical approval code 10619.®) at 3 mg/kg; IM xylazine HCl (Rompun®) at 2.5 mg/kg and IM midazolam (MidazolamGen®) at 0.17 mg/kg. Afterward, anesthesia was induced with IV propofol (Propofol Lipuro 1%®) at 2.5 mg/kg from Boehringer Ingelheim, Bayer AG, GES and BBraun, respectively. Maintenance for 6 h was achieved with inhaled isoflurane (Forane® 2%) of Centauro Veterinary administered by tracheal intubation with a low-pressure balloon, number 7.Thirteen female pigs (Yorkshire-Landrace) of 45–50 kg were anesthetized for 6 h with a standard sedation protocol based on IM ketamine  of animal investigation.One female pig was not administered anything (white). CP-Solution was administered to six female pigs and CP-NP was administered to six female pigs . Each formulation (CP-Solution and CP-NP) was administered topically and locally in each studied tissue  in pigs. The same formulation (CP-Solution or CP-NP) was administered in the tissues studied in the same pig. In summary, Qr) of the drug.After the in vivo permeation phase, studied porcine mucous and ophthalmic tissues were obtained immediately after the animals were sacrificed by an intravenous overdose of sodium thiopental. Samples were collected and fixed overnight (ON) by immersion in 4% paraformaldehyde (PFA) in phosphate-buffered (PB) 20 mM, pH 7.4 and further processed for paraffin embedding. Vertical histological sections were obtained, stained with hematoxylin and eosin and mounted under a cover slip to be observed at 400× and photographed with a Leica DMD 108 optical microscope. In addition, part of the original tissue was used to calculate the in vivo retained amount . The resulting solutions were measured by HPLC–UV yielding the amount of CP retained in the membrane .At the end of the in vivo study, the mucous membrane specimens were used to determine the amount of retained drug. Mucous and ophthalmic samples were carefully cleaned with gauze soaked in a 0.05% solution of sodium lauryl sulfate, washed with distilled water and blotted dry with filter paper. The permeation area was then excised and weighed. Later, different tissues were perforated using a 30 G needle . Its CP content was extracted with methanol/buffer phosphate solution in an ultra-sonic processor for 20 min .Histological studies of all the tissues have been carried out in order to verify if the formulations studied do affect the tissue structure and therefore, or its effect is attributable to the drug itself. In all tissues, a blank histological study (without drug), a CP-Solution histological study and a CP-NPs histological study have been performed . The mucous membranes are composed of two parts: epithelium (A) and connective tissue (B). Inside the epithelium, the outermost part is the stratified flat keratinized epithelium (A) and the connective tissue is its own laminate (B). These two parts are separated by the basal layer. This is the basic structure of the mucous membranes and each tissue presents its own particularities detailed in the photographs.Js) is statistically higher in the CP-Solutions (9.4 mcg/h) than in CP-NPs (4.19 mcg/h). This is interesting, since the flow is the rate of entry into the eye, preventing its residence. So, in this type of mucous, the NPs stay longer in the tissue that is the place of action of the drug. In addition, the Tl of the NPs is statistically lower (0.31 h) compared to the results of the CP-Solution (1.45 h). Tl indicates the time required to reach a steady state, therefore, the results suggested that CP-NPs are absorbed very rapidly in this tissue and had a high diffusion [Kp, it is statistically higher in the CP-Solution (19.58 cm h) than in the CP-NPs (8.73 cm h). The Kp depends a lot on the formula of the drug, and in this case, the NPs have lower permeability, they stay longer in the tissue and can perform their function. Therefore, this may be an indication that NPs can have more effect for a longer time [P1 and P2 between the two types of formulations. P1 indicates the distribution between the formulation and the tissue and P2 indicates the diffusion of the drug into the tissue. In this case, P1 is statistically higher in the CP-Solution formulation (17.01 cm) than in the CP-NPs (2.18 cm). In other words, the distribution between vehicle and membrane is greater in CP-Solutions, and therefore, it penetrates better than CP-NPs. An explanation would be the product formulation itself, since the CP-Solution is dissolved and the CP-NPs are encapsulated [P2 is statistically higher in the case of CP-NPs (6.32 cm). These results show that although NPs have a shorter lag time penetrating the tissue (probably due to their formulation) [Qr of the two types of formulations (3.62 mcg/cm2/g in CP-Solution and 3.57 mcg/cm2/g in CP-NPs). In summary, in the conjunctive mucous, we can say that CP-NPs have advantages, since they have a lower Js and Kp and a higher P2 than the CP-Solution. Therefore, CP-NPs have a lower permeation capacity and a better diffusion inside the tissue.As we can see in iffusion . In the ger time . There alution 1. h. Tl inulation) , once inQr. So, both formulations act in a very similar way. Although there are no differences between J and Kp, the Qr of the CP-NPs (20.89 mcg/cm2/g) is statistically greater than in the CP-Solution (16.56 mcg/cm2/g). Therefore, we can affirm that CP-NPs are retained in the mucous and therefore have more local activity and are safer (since less drug passes into the bloodstream).In cornea, no statistically significant differences are observed for any parameter , except Js and the Kp between formulations  than the CP-NPs (2.7 h−1). In addition, the CP-Solution has a statistically higher P2 (0.82 cm) than the CP-NPs (0.62 cm). These two parameters suggest that in this tissue the CP-Solution has advantages over the CP-NPs, since CP-Solution takes less time to penetrate the tissue and also diffuses better inside the mucous membrane.In the sclera, there are no significant differences between the ulations . Only stIn brief, in the eye, we can conclude that CP-NPs have advantages in the conjunctiva and cornea. In the conjunctiva, CP-NPs take longer to penetrate, so they stay longer in the place of action. In the case of the cornea, although the two formulations act in a similar way, the CP-NPs have a better local activity and are safer. So, CP-NPs are a good option to treat locally inflammatory diseases in these two tissues.Kp are statistically lower in the CP-Solution (0.74 mcg/h in CP-Solution and 0.39 cm in CP-NPs). Therefore, CP-Solution has a lower capacity for permeation and less entry into the bloodstream. However, other factors such as Tl, P1, P2 and Qr should be commented on and considered. Tl is statistically higher in the CP-Solution (1.65 h) than in the CP-NPs (0.8 h). In addition, P1, P2 and Qr are statistically significantly higher in CP-NPs. A very important factor to consider is that Qr in CP-NPs is statistically higher (3.46 mcg/cm2/gr) than in CP-Solution (2.38 mcg/cm2/gr). Therefore, we can say that CP-NPs are safer and more efficient to treat buccal mucous membranes.As we can see in J) is statistically lower in the CP-NPs (0.31 mcg/h) than in the CP-Solution (4.81 mcg/h)  and this factor also makes it a better treatment locally in this tissue. Another factor to take into account is that the Tl of the CP-NPs is statistically lower (0.8 h) than that of the CP-Solution (2.77 h), and therefore, it is absorbed into the tissue more quickly. Significant differences are also observed in P1 and P2. In this case, the P1 is statistically higher in the CP-Solution (16.24 cm); but, the P2 is statistically higher in the CP-NPs (2.1 h−1) than in the CP-Solution (0.6 h−1). Therefore, CP-NPs spread faster inside the sublingual mucous. However, a negative factor is that in this tissue the Qr is slightly statistically higher in the CP-Solution (33.14 mcg/cm2/g) than in the CP-NPs (29.13 mcg/cm2/g). This factor is important, since the higher the Qr, the more effective and safer the drug is. However, although the Qr is significantly higher in the CP-Solution, the amount of Qr in the CP-NPs is also quite high. In summary, taking into account all the factors, we can affirm that CP-NPs are a good candidate for the local treatment of inflammatory diseases.In the sublingual mucous, it can be observed that the flow (1 mcg/h) . TherefoJ) and Kp are statistically lower in the CP-Solution  than in the CP-NPs . Therefore, permeation into the bloodstream is lower in the CP-Solution. However, Tl is statistically lower in the CP-NPs (1.75 h) than in the CP-Solution (3.34 h). Then, CP-NPs reach a steady state faster than CP-Solution. In addition, P2 is statistically higher in CP-NPs (0.99 h−1) than in CP-Solution (0.53 h−1). So, the diffusion of CP-NPs within the tissue is better. Finally, we can see that the Qr in the CP-NPs is much higher (52.81 mcg/cm2/g) than in the CP-Solution (23.44 mcg/cm2/g). Therefore, the amount of drugs retained in vaginal tissue is higher in CP-NPs, and ultimately, they are safer and more effective.The last tissue to consider is vaginal mucous . In thisIn summary, we can affirm that CP-NPs have particular advantages in these three tissues. In the sublingual mucous, CP-NPs take longer to penetrate, so they stay longer in the place of action. In addition, CP-NPs are safer and more effective in the buccal and vaginal mucous. So, CP-NPs are a good option to treat locally inflammatory diseases in these three tissues.When reviewing histological photos, it is important to look at and compare several parameters. The most important part to analyze is the areas that have been in contact with the drug. Therefore, we will focus on the outermost area of the mucous (stratified flat keratinized epithelium and own laminate). On the other hand, it is also important to verify that the cellular structures that make up each tissue do not show alterations ,48. ThenIn buccal mucous  we can sSublingual mucous tissue is a much more muscular tissue than the rest (C) and with much more collagen (D) . HoweverWe can see vaginal mucous tissue presents undulations . This isWe can see the structure of the cornea in As we can see in In this last figure, we can see the structure of the sclera . This tiDefinitely, no histopathological or significant structural changes were observed between control and treated samples. The epithelial cells and the connective tissue beneath showed normal morphology and distribution in the histological mucosal samples analyzed. These results strongly suggest that CP-Solution and CP-NPs do not affect the cellular and tissular morphology and organization in locally in vivo treatment.In conclusion, the results show that CP-NPs have advantages in most tissues. They are more effective and safer than the CP-Solution and do not alter the tissue structure. This presents great possibilities for the local treatment of many inflammatory diseases in pigs or humans. In this approach, the side effects of NSAIDs will be minimized. However, additional studies are required to formulate a pharmaceutical presentation that will be easier to apply in pigs to facilitate its administration and animal management.


Replacing occupational sitting time with active tasks has several proposed health benefits for office employees. Mobile phones and motion sensors can provide objective information in real time on occupational sitting behavior. However, the validity and feasibility of using mobile health  devices to quantify and modify occupational sedentary time is unclear.The aim of this study is to validate the new Walk@Work-Application (W@W-App)—including an external motion sensor (MetaWearC) attached to the thigh—for measuring occupational sitting, standing, and stepping in free-living conditions against the activPAL3M, the current gold-standard, device-based measure for postural behaviors.t tests and Wilcoxon signed-rank tests. Agreement between measures was examined using concordance correlation coefficients (CCCs), 95% CIs, Bland-Altman plots , and equivalence testing techniques.In total, 20 office workers  downloaded the W@W-App to their mobile phones, wore a MetaWearC sensor attached to their thigh using a tailored band, and wore the activPAL3M for 3-8 consecutive working hours. Differences between both measures were examined using paired-samples P<.001). No significant differences between sitting (P=.53), standing (P=.12), and stepping times (P=.61) were identified. The CCC identified substantial agreement between both measures for sitting , moderate agreement for standing , and poor agreement for stepping . Bland-Altman plots indicated that sitting time  and standing time  were underreported. For stepping time, a positive mean bias of 1.15 minutes (95% LoA –15.11 to 17.41) was identified. Equivalence testing demonstrated that the estimates obtained from the W@W-App+MetaWearC and the activPAL3M were considered equivalent for all variables excluding stepping time.The median recording time for the W@W-App+MetaWearC and the activPAL3M was 237.5 (SD 132.8) minutes and 240.0 (SD 127.5) minutes, respectively (The W@W-App+MetaWearC is a low-cost tool with acceptable levels of accuracy that can objectively quantify occupational sitting, standing, stationary, and upright times in real time. Due to the availability of real-time feedback for users, this tool can positively influence occupational sitting behaviors in future interventions.ClinicalTrials.gov NCT04092738; https://clinicaltrials.gov/ct2/show/NCT04092738 Replacing sedentary time  . PositivSelf-monitoring is a key element to increase individuals’ awareness of and empowerment toward behavior change . For PA Evidence has identified mobile phones as a potential alternative to accurately self-monitor PA and SB via inbuilt inertial sensors -15. HoweCommercially available devices that examine SB through postural positioning rather than lack of movement  are scarcer . HoweverMobile phones alone struggle with postural identification due to the nonattachment of phones to the body and the ubiquitous nature of phone use . HoweverIn this context, the Walk@Work-Application (W@W-App) was developed to self-monitor occupational PA and SB with a high level of validity. The W@W-App communicates with a MetaWearC external sensor , attacheThe new W@W-App was developed from a previous version , adding The algorithm for the W@W-App+MetaWearC  was desiThe recognition of postures (sitting and standing) is based on the angle of the z-axis, where 0 indicates a completely vertical posture (standing) and 1 indicates a completely horizontal posture (sitting). When the sensor detects a value higher than 0.8 in the z-axis, the sitting counter is initiated while the standing counter remains to 0. When the sensor detects a value equal to or lower than 0.8 in the z-axis, the standing counter begins while the sitting counter returns to 0. If either the sitting or standing counters reach 75 readings (approximately every 2 seconds), this indicates that the sensor has not detected stepping during those 75 readings, and therefore the step counter stops and assumes that the user is either sitting or standing depending on which of these counters reaches 75.Finally, if there is a difference greater than 15 minutes between the time counters for the W@W-App-MetaWearC  and the elapsed time, a weighted adjustment is completed. Normally, this difference is due to temporal disconnections of the sensor if it is kept more than 20 meters away from the mobile phone. For example, if the W@W-App-MetaWearC has counted for 100 minutes , but the real time elapsed is 115 minutes, the weighted adjustment will correct the W@W-App-MetaWearC to 86 minutes stepping, 12 minutes standing, and 17 minutes sitting.The activPAL3M (PAL Technologies Ltd) is referred to as the gold-standard, device-based measure for postural recognition in free-living conditions . The actOffice workers from the University of Vic-Central University of Catalonia (UVic-UCC) who owned a mobile phone (Android version 6.0.0/iOS version 10.0.0 or higher) were invited to participate in the study. A convenience sample was recruited (N=23). All volunteers provided written informed consent prior to participation. This study was conducted within a Spanish national project  led by the UVic-UCC. Ethical approval was obtained by the research ethics committee of the Research Institute of Primary Care Jordi Gol (IDIAP).Participants installed and configured the W@W-App, following guidance provided by the researchers: (i) registration on the Walk at Work web platform , (ii) usResearchers initialized the activPAL3M and the W@W-App+MetaWearC and placed both devices on the midpoint  of the anterior aspect of the thigh of the same leg to avoid measurement bias due to asymmetric leg positions and movements. To ensure that the timestamp of the W@W-App+MetaWearC and the activPAL3M aligned for data analysis, they were initialized from the same PC.Participants wore the W@W-App+MetaWearC and the activPAL3M sensor in occupational free-living conditions for 3-8 hours. They were required to keep their mobile phone within a 5-meter radius throughout the measurement period .The variables recorded and quantified by the W@W-App+MetaWearC were time spent in sitting and standing postures and time spent stepping. These variables were extracted from the W@W-App software. For the activPAL3M, files were processed via the activPAL Professional Software (version 7.2.32) upon completion of data collection. Data were then exported to a Microsoft Excel  file format, providing data on sitting, standing, and stepping in 15-second epochs. This enabled the quantification of the number of minutes spent sitting, standing, and stepping. In addition, the time spent sitting and standing were added together to quantify stationary time, while the amount of time spent standing and stepping were added together to compute upright time. Total recording time  from both devices was calculated by summing the amount of time spent sitting, standing, and stepping.t tests and Wilcoxon signed-rank tests. Pearson correlation coefficients were used to determine the strength and direction of association between variables quantified by the two measures when the data were normally distributed. Spearman rank-order correlation coefficients were employed when data were not normally distributed. The concordance correlation coefficient (CCC), using Lin’s approach [t tests (90% CI) for the mean difference between the W@W-App+MetaWearC variables and the activPAL3M determined variables [Descriptive characteristics (mean [SD] and median [IQR]) were used to describe the data. Differences between the W@W-App+MetaWearC and the activPAL3M were examined using paired-samples approach , was useapproach . Bland-Aapproach . Equivalariables . Equivalariables . MeasureIn total, 23 office workers participated in the study, whereby activity behavior information was recorded by both measures during workplace free-living conditions between October and November 2018. After excluding 3 participants because of technical problems with the mobile phone, data from 20 participants were included in the analyses . A total of 115 hours of data was recorded, with an average of 5 hours per participant. Of all participants, 13 used an Android smartphone  with an operational system version ranging from 6.0.1 to 8.0.0. The other 7 participants employed an iPhone 6 or iPhone 7 with an operational system version higher than 10.3.3.P=.53), standing time (P=.12), and stepping time (P=.54).Descriptive characteristics for variables of interest from the W@W-App+MetaWearC and the activPAL3M, as well as the statistical differences between the two measures for each variable, are described in The W@W-App+MetaWearC showed strong to very strong correlations with activPAL3M-determined activity variables. CCCs identified substantial agreement between the two measures for sitting , moderate agreement for standing , and poor agreement for stepping . The correlation coefficients, CCC values, and associated 95% CI are shown in The mean bias and LoA from the Bland-Altman analysis are provided in P=.002), while no differences were observed for upright time (P=.25). However, stationary and upright times were strongly correlated with the criterion measure (activPAL3M) (P<.001). Time spent on stationary activities was underestimated with a mean bias of –6.52 minutes, with a relatively small LoA (–20.81 to 7.78 minutes). The 90% CI for the mean difference was 1.3 and 12.4 and was within the 15% equivalence region (–41 to +41 minutes). The estimates obtained from the two measures were considered equivalent for stationary time. A mean bias of –1.85 minutes was identified for time spent upright, with a relatively small LoA (–15.76 to 12.06 minutes) compared to the noncombined postural/activity variables. The equivalence procedure indicated that the 90% CI for the mean difference was –0.9 and 4.8 and was within the 15% equivalence region (–7.0 to +7.0 minutes). The estimates obtained from the two measures were considered equivalent for upright time.When combining variables, stationary time significantly differed between the two measures . However, the analysis demonstrated that the findings for stepping from the W@W-App+MetaWearC are not equivalent to those from the activPAL3M.Although a small mean bias of 1.15 minutes for stepping was observed between the W@W-App+MetaWearC and the activPAL3M, poor agreement, wide CIs and nonequivalence would suggest that the W@W-App+MetaWearC should not be recommended for use in detecting stepping time. However, it is quite plausible that these observations can be attributed not only to variance from the W@W-App+MetaWearC tool but also variance in the activPAL3M device. The activPAL3M is primarily used to examine postural position, namely sitting and standing, and has demonstrated high levels of accuracy in the detection of these behaviors in lab-based and free-living conditions , justifyFor stationary time , the W@W-App+MetaWearC demonstrated high levels of accuracy when compared with previous validation studies employing a range of activity monitors . This isThe W@W-App+MetaWearC is a novel tool that simulates the activPAL3M activity monitor in accurately recognizing postural position at the workplace. The output from the W@W-App+MetaWearC tool for sitting, standing, stationary, and upright times were identified as equivalent to the current gold-standard, device-based postural measure, the activPAL3M. This suggests that this self-monitoring tool, which provides real-time feedback to users, is worthwhile for use in interventions that aim to reduce sitting behavior in the workplace. Self-monitoring is vital for increasing individuals’ awareness and empowerment toward behavior change . This maThe strengths of this study include (i) the examination of the complete range of occupational sedentary and activity behavior types , (ii) the examination of the validity of these measures in occupational free-living conditions, and (iii) the use of a gold-standard, objective measurement device to determine the validity of the W@W-App+MetaWearC.The present study is not without limitations. Although the activPAL3M has been described as the gold standard for measuring sitting time  and is aThe W@W-App+MetaWearC self-monitoring system demonstrates high levels of accuracy in determining postural position. This tool is a low-cost alternative tool for the examination of occupational sitting and standing times. It has demonstrated high levels of validity in detecting postural position and provides real-time feedback to users. Future research should examine the interventional effect of utilizing this system as a self-monitoring tool for modifying activity behaviors in office-based workers.


Pisum sativum and other legumes. While the enzymes responsible for the halogenation of compounds in bacteria and fungi are well studied, the metabolic pathways leading to the production of 4-Cl-IAA in plants, especially the halogenating reaction, are still unknown. Therefore, bacterial flavin-dependent tryptophan-halogenase genes were transformed into the model organism Arabidopsis thaliana. The type of chlorinated indole derivatives that could be expected was determined by incubating wild type A. thaliana with different Cl-tryptophan derivatives. We showed that, in addition to chlorinated IAA, chlorinated IAA conjugates were synthesized. Concomitantly, we found that an auxin conjugate synthetase (GH3.3 protein) from A. thaliana was able to convert chlorinated IAAs to amino acid conjugates in vitro. In addition, we showed that the production of halogenated tryptophan (Trp), indole-3-acetonitrile (IAN) and IAA is possible in transgenic A. thaliana in planta with the help of the bacterial halogenating enzymes. Furthermore, it was investigated if there is an effect (i) of exogenously applied Cl-IAA and Cl-Trp and (ii) of endogenously chlorinated substances on the growth phenotype of the plants.Plant hormones have various functions in plants and play crucial roles in all developmental and differentiation stages. Auxins constitute one of the most important groups with the major representative indole-3-acetic acid (IAA). A halogenated derivate of IAA, 4-chloro-indole-3-acetic acid (4-Cl-IAA), has previously been identified in  The halogenated auxin 4-chloro-indole-3-acetic acid (4-Cl-IAA) is among the strongest auxins identified in plants up to date . It stimPisum sativum (pea) [Medicago truncatula and Trifolium repens [Evidence for the occurrence of 4-Cl-IAA was first found in germinating seeds of um (pea) ,8 by detum (pea) ,10 and tum (pea) ,12,13. Ium (pea) , surfaceum (pea) . There aum (pea)  found thum (pea) . Chlorinm repens . HoweverHalogenated compounds occur widely in nature. Today more than 5000 naturally produced halogenated substances are known and their number is increasing . The strCatharanthus roseus hairy roots the existence of new halogenated compounds with modified properties after the incorporation of simple halogenated precursor was shown [Arabidopsis thaliana, and it was shown that these plants contained chlorinated Trps as well as IAA [A. thaliana. Furthermore, we have evaluated the capacity of these transgenic lines to produce chlorinated Trp and other indole derivatives. In addition, wild type plants were incubated with different halogenated compounds to test the effect of the substances on plant growth. Such investigations have so far not systematically been carried out for A. thaliana with Trp and IAA derivatives chlorinated in most possible positions on the indole ring. Finally, the expression patterns of selected genes encoding proteins from the IAA synthesis pathway involving indole-3-acetonitrile (IAN) have been studied.To show the potential of chlorinated compounds synthesized by plants, different strategies have been used. Employing as shown . More rel as IAA . In thisA. thaliana plants. Both Trp and IAA derivatives were chlorinated in different positions on the indole ring and compared to non-chlorinated compounds  is possible in transgenic a plants . To get ompounds . A. thaliana used a broader range of amino acids when 7-Cl-IAA was the substrate  produced heterologously in no acids  despite s of IAA . The cons of IAA . It is ps of IAA . Up to nA. thaliana plants after the stable transformation with the Trp 5-halogenase gene pyrH, as well as for the plant codon-optimized gene pyrHopt without any co-introduction of a flavin reductase [pyrH gene from Streptomyces rugosporus, other halogenase genes  [A. thaliana via floral dip [pyrH was higher expressed in the two lines chosen compared to four lines of the non-codon-optimized version. Although the different expression level might also be due to positional effects of integration, it could also be the result of a better transcription of the optimized version. However, this difference does not correlate with the levels of Cl-Trp   were comrescens)  and exproral dip . Expressf Cl-Trp  and therf Cl-Trp . We therA. thaliana lines containing the six different constructs are summarized. We found, in all cases, chlorinated Trp as well as chlorinated IAN. The amount of 5-Cl-Trp varies in the range of 1.7 to 5.15 µg g−1 lyophilized tissue  [−1) than the overexpressor line (8–12 µg), indicating that the Cl-Trp was further converted via Cl-IAN to other compounds, maybe IAA.To determine whether S::NIT2)  and incuThe different transgenic lines shown in A. thaliana, which has been achieved for many transgenic lines.Neither the number of rosettes/leaves nor the rosette diameter is significantly altered in comparison to the wild type plants and to other transgenic lines A. When wA. thaliana resulted in functional enzymes by the formation of Cl-Trp as well as a possible intermediate of the IAA pathway, Cl-IAN, and Cl-IAA. We wanted to analyze whether the presence of Cl-IAA in transgenic plants could have an effect on the expression of genes encoding enzymes of the Trp-dependent IAA synthesis pathway involved in the formation of IAN as an intermediate compared to the wild type. To possibly stimulate the gene expression of the pathway we also added either Trp or 5-Cl-Trp at two different concentrations  or only after treatment with 5-Cl-Trp (CYP79B2), while NIT2 expression was mainly upregulated in this line after treatments with Trp and Cl-Trp. However, the second line harboring a non-optimized construct showed a different pattern, namely upregulation of CYP79B2 and NIT2 by high concentrations of Cl-Trp, but CYP79B3 and NIT1 were either not regulated at all or, in one case, downregulated. In line AtpyrHopt OX_6 there was an upregulation of CYP79B2 for one and CYP79B3 for all Cl-Trp treatments, while only NIT2 expression was upregulated by both Trp derivatives at high concentrations. In conclusion, upregulation of NIT transcription in some lines, including wild type, by Trp and Cl-Trp (at different concentrations) could indicate a role for this enzyme in the conversion of high levels of precursor and also, together with the CYP79B2/B3 genes, in stress response , also has a growth promoting effect on A. thaliana [A. thaliana roots grown on 4- and 5-Cl-IAA behaved similarly  with all four tested Cl-Trp derivatives as substrates. A. thaliana conjugates of IAA and Asp are thought not to be a conjugate for the storage of IAA, but a precursor for degradation [In feeding experiments we were able to show not only the uptake of chlorinated Trp but also its metabolism. The conversion of Cl-Trp variants that have the Cl-atom at different positions on the indole ring to the respective chlorinated indole derivatives in radation ,49. One radation ,51. No oradation  using chradation  who coulpyrH and pyrHopt we were able to demonstrate such a difference in favor of the transcription of the latter. For all other constructs and the resulting gene expression values, only individual differences between individual lines were determined. These disparities within a set of lines harboring the same bacterial halogenase gene could have different reasons. One could be the integration of the halogenase genes into different sites within the genome of A. thaliana. Furthermore, an increased expression does not necessarily lead to an increased accumulation of Cl-Trp. In our studies it was not possible to analyze the amounts of enzymes present in the individual lines, so we can only draw conclusions from the formation of the chlorinated product Trp. In A. thaliana, Trp is the precursor of many different metabolites such as IAN, IAA [The optimization of bacterial genes should lead to their better transcription in a plant system and ultimately enable a stronger expression. For other expression systems, it was possible to show that the optimization of genes for the corresponding organism leads to an increased expression and increased formation of enzymes ,54. HoweIAN, IAA , camalexIAN, IAA  and indoIAN, IAA . To whatFor different bacterial halogenases  it was shown in vitro that these enzymes are enabled to halogenate IAA directly . However−1 of Cl-Trp and about 0.2 µg·g−1 Cl-IAN, each based on the dry weight. If a conversion from Cl-IAN to Cl-IAA had taken place, it could be possible that this was not detected due to the low concentrations. As the possibility exists that small amounts of IAA and Cl-IAA are degraded under light, we used yellow protective covers for these experiments to minimize that effect. As shown before, IAA and Cl-IAA were stable in solid and liquid medium over a period of 17 days, as about 80% of the compounds were still detectable [A. thaliana towards modified substrates. Preliminary experiments point to the possible conversion of 5-Cl-Trp to downstream metabolites to a higher extent than wild type by a nitrilase overexpressing line . So farA. thaliana specific pathway via IAN, via which we detected the chlorinated compounds. We therefore analyzed the expression of two cytochrome P450-dependent genes encoding enzymes of the first committed step to indole glucosinolates/IAA (CYP79B2/B3) [NIT1/2) encoding the enzymes responsible for the formation of IAA from IAN [pyrH and a plant expression-optimized version of the same gene. Consequently, the incubation was done with the 5-Cl derivative. However, only line-dependent alterations in gene expression were detected. Therefore, we cannot rule out that some of the effects observed could be due to the known stress response of some pathway genes resulting in altered gene expression [When a substrate of a biosynthetic pathway is added, this usually results in higher levels of product, but we also wanted to know whether chlorinated compounds affect the expression of genes encoding enzymes for the 79B2/B3)  and alsofrom IAN ,59. The Arabidopsis thaliana (L.) HEYNH. ecotype Col-0 was used as the wild-type controls as well as for generating transgenic plants overexpressing the different halogenase genes  resulting in lines AtpyrH, AtpyrHopt, AtthdH, AtthdHopt, AtprnA, AtprnAopt. To generate the transgenic plants the original and optimized (opt) genes  were amplified using PCR with specific primers  [ primers  and inseltration  into theS::NIT2) .A. thaliana plants were grown under greenhouse conditions  or sterile in petri dishes and liquid cultures in plastic cups . The sterile cultivation for selection (with 50 µg hygromycin), the growth tests and the feeding experiments were performed with standard MS medium containing 1% sucrose, pH 5.8. For the petri dish tests 1.5% phyto-agar was added together with the appropriate concentrations of chlorinated Trp or auxin derivatives . The seeds were sterilized by treating them with 75% ethanol for 5 min, followed by 15 min in a sodium hypochlorite solution and four wash steps in sterile distilled water. The seeds were stratified in the dark at 4 °C for two days. The incubation was performed in growth chambers at 23/18 °C under a 16/8 h light/dark period for 17 days or in the greenhouse on a shaker (60 rpm). We chose this time point because (i) the same was used for the other analyses where we needed to collect enough material and (ii) because prominent differences were only found at such later time points. To protect IAA and Cl-IAA from degradation, yellow plastic shields were used [The ere used .For the routine greenhouse cultivation and the phenotype analysis the plants were placed after 10 days on the selection media in pots with soil and sand (3:1). The phenotypic studies of the plants were done following Boyes et al. . For eveAtYLS8 (AT5G08290.2) as control.The plants were incubated in liquid MS culture under sterile conditions for 14 days and treated for 24 h in the corresponding Trp- or 5-Cl-Trp-containing medium. The plant material was mortared with liquid nitrogen and RNAzol  directly after harvesting. After being mortared, the plant material samples were stored at −80°C until RNA extraction. The RNA extraction was performed following the manufacturer instructions. In addition to this a DNA removal step was added, to get DNA-free RNA. The digestion of the DNA was performed with DNase I  and 1–20 µg RNA. For the quantitative real-time PCR (qRT-PCR) the cDNA was synthesized with Maxima First Strand Reverse Transcriptase  following the manufacturer information with 1 µg RNA. The SensiFAST SYBR No-Rox Kit by Bioline was used for qPCR as described by the manufacturer. All reactions were performed as triplicates with the following conditions: 95 °C for 5 min, 40 cycles each at 95 °C for 20 s and 62 °C for 20 s, and 72 °C for 20 s. After the cycles a melting curve was performed to confirm that the correct product was synthesized. Specific primers used for qRT-PCR are shown in A. thaliana plants, as well as the wild type plants which were incubated with the chlorinated Trp derivatives, were done as described in Patallo et al. [2 = 0.9972), chlorinated IAAs , IAA  and IAN  and the curves were used for quantification. Since standard compounds for each of the IAA conjugates were not available, we used the normalized peak areas as a relative measure of the abundance of the compounds.The analysis of possible chlorinated products in the plant extracts of the transgenic o et al. . In botho et al. . Multiplo et al. , and theGH3.3 gene from A. thaliana was PCR-amplified from cDNA essentially as described for the cloning of Physcomitrella patens GH3 genes and cloned into the same expression vector as described in [E. coli BL21 cells, and for the purification of the enzyme we used an IPTG inducing expression plasmid with a glutathione tag. For the purification we applied the protein onto a glutathione-sepharose matrix. An overnight culture of the cells  was centrifuged for 10 min . The pellet was resuspended in 250mL standard LB media, but without glucose , and IPTG (1 mM) was added. The culture was incubated for 3–4 h . The cells were centrifuged again  and washed with 2 ml TB buffer. The resulting pellet was frozen in liquid nitrogen. The purification with the glutathione-sepharose matrix was carried out according to the manufacturer’s specifications . The elution of the GST-fusion protein was done for 10 min with 30 µl elution buffer (GB buffer). The concentration of the eluted protein was analyzed with SDS page and BSA standards. For the enzyme assay, IAA, 4-Cl-IAA, 5-Cl-IAA, 6-Cl-IAA and 7-Cl-IAA in a concentration of 1mM were used as substrates. The reaction mixture consisted of reaction buffer 1×, ATP 3 mM, DTT 1 mM, amino acids 1 mM and 2.5 µg purified protein. The reaction mixture was incubated overnight at 25 °C, frozen in liquid nitrogen and stored at −10 °C until analysis.Whether the formation of chlorinated IAA conjugates with the enzyme AtGH3.3 is possible was investigated with an enzyme test. The ribed in . The vecThe formation of possible chlorinated IAA conjugates from the enzyme assay was subsequently analyzed in more detail using TLC. The free and conjugated IAA were separated for 15 min on Silicia Gel F254 plates in the solvent mixture of chloroform:ethyl acetate:formic acid (35:55:10). The focus was not on the quantification of the samples, but on the formation of possible conjugates with Cl-IAA. Nevertheless, uniform amounts of starting material were applied. The conjugate formation was visualized with Van Urk-Salkovski reagent. The dried plates were sprayed with the reagent mixture and incubated for 10 min at 60 °C .p < 0.05 or p < 0.01 level.The ANOVA analysis was done using SPSS software . Statistically significant differences were indicated at A. thaliana is possible and that this can result in the regioselective formation of chlorinated Trp derivatives depending on the type of halogenase produced. In addition to Cl-Trp, we have shown that the synthesis of metabolites from the IAA biosynthetic pathway, namely Cl-IAN, and the auxin itself, Cl-IAA, starting from Cl-Trp in A. thaliana is possible. In addition, an increased efficacy of chlorinated compounds (both Trp and IAA) on the growth of wild type plants has been demonstrated. Formation of halogenated compounds, however, did not lead to phenotypic changes in the transgenic plants. Our results provide the basis to use these strategies in the future in other A. thaliana lines with altered spectra of metabolites of the pathway to produce higher amounts of the more bioactive chlorinated auxins or other compounds derived from the Trp moiety, i.e., camalexin or indole glucsoinolates.While the native enzyme converting Trp to the Cl-derivative in pea until today is not known, we have successfully shown that heterologous expression of different bacterial halogenase genes in


Sensitivity to acetylsalicylic acid (ASA) is important in the treatment of patients with coronary heart disease (CHD) after coronary artery bypass grafting (CABG). Patients were divided into ASA sensitive (sASA) and ASA resistant (rASA) by the activity of platelet aggregation induced arachidonic acid (ARA) together with ASA. Induced platelet aggregation activity was studied in sASA and rASA patients with CHD before and after CABG. The level of synthesis of primary and secondary reactive oxygen species (ROS) by platelets was determined using chemiluminescent analysis. The activity of NAD- and NADP-dependent dehydrogenases in platelets was determined by the bioluminescent method. It was found that the aggregation activity of platelets depended on the sensitivity of CHD patients to ASA and decreased during postoperative ASA therapy. The most pronounced differences in metabolic parameters of platelets in sASA and rASA patients were detected by Nox2 activity. The synthesis of secondary ROS by platelets of CHD patients did not depend on the sensitivity of patients to ASA but increased during postoperative treatment with ASA. The activity of NAD(P)-dependent dehydrogenases in platelets did not differ in sASA and rASA patients with CHD. Acetylsalicylic acid (ASA) is the most widely used drug for the prevention of cardiovascular events including adverse outcomes in coronary heart disease (CHD). In addition, ASA is used to reduce the risk of shunt occlusion after coronary artery bypass grafting (CABG) ,2. ASA aWe also believe that the functional activity of platelets is largely determined by the state of their metabolism. Accordingly, ASA resistance may also depend on platelet metabolism. It was proven that the functional activation of platelets was carried out with enhanced synthesis of reactive oxygen species (ROS) by enzymes that are located on the outer cytoplasmic membrane and inside the cells (including in mitochondria) ,10. The Plastic and energy metabolism also contribute to the state of the functional activity of platelets. Plastic reactions determine the synthesis of surface platelet receptors and various humoral factors, while energy metabolism supplies energy for these processes ,22. It hHowever, the role of metabolic processes (including synthesis of ROS) in the implementation of the functional activity of platelets in CHD patients with different sensitivity to ASA has not yet been studied. It is important to note that the functional activity of platelets can change after CABG, which is associated with the use of extracorporeal circulation (by activating platelets upon contact with synthetic surfaces) and subsequent postoperative therapy (in particular the use of ASA) of patients ,27,28. FWe chose the activity of NAD- and NADP-dependent dehydrogenases as the studied indicators of platelet metabolism based on the following. Firstly, pyridine nucleotides (coenzymes of dehydrogenases) are the main carriers of electrons in cells and, therefore, these enzymes take an active part in energy processes ,30. Secop < 0.001) and persisted for one (p < 0.001) and 8–10 (p = 0.006) days after CABG. The platelet aggregation on sASA patients with CHD before and after CABG was lower than in patients of the control group. Higher level of the platelet aggregation compared with control values was detected on rASA patients before CABG (p < 0.001) and the first day after CABG (p = 0.042). ARA-Induced platelet aggregation with the addition of ASA on patients of this group on the 8–10th day after CABG corresponded to control values.The result of platelet aggregation (with various inductors) measured on CHD patients with ASA sensitivity (sASA) and ASA resistance (rASA) before and after CABG are presented in p = 0.010). ARA-Induced platelet aggregation in sASA patients on the 1st day after CABG decreased relative to control (p < 0.001) and initial (p < 0.001) values and also became lower than in rASA patients (p = 0.027). rASA Patients in this period also had a lower level compared with the control (p < 0.001) and the initial (p = 0.012) values of the ARA-induced platelet aggregation. Similar ratios of ARA-induced platelet aggregation on sASA and rASA patients were also observed eight to 10 days after CABG.There was no significant difference in the ARA-induced (no ASA in samples) platelet aggregation between sASA and rASA patients with CHD before CABG b. Patienp < 0.001 with all comparisons) .ADP-induced platelet aggregation in sASA and rASA patients with CHD was lower than in control subjects during the entire observation period (arisons) c. There p = 0.013 and p < 0.001, accordingly) and eight to 10 (p < 0.001 in both cases) after CABG. rASA Patients on the first day after the CABG had a reduced level of the aggregation relative to the initial level (p = 0.019) whereas on the 8–10th day a decrease was revealed relative to the control and initial values . There were no differences in the levels of collagen-induced platelet aggregation in sASA and rASA patients after CABG.The level of collagen-induced platelet aggregation in CHD patients before CABG did not differ from control values and there were no differences between sASA and rASA patients d. The lep < 0.001). Patients with sensitivity and ASA resistance on the 8-10th day after the CABG also had a decrease in the levels of adrenaline-induced platelet aggregation relative to the initial values (p < 0.001 in both cases). Differences in the levels of adrenaline-induced platelet aggregation between sASA and rASA groups of patients with CHD before and after CABG were not detected.Levels of adrenaline-induced platelet aggregation in sASA and rASA patients with CHD before and after CABG were reduced relative to control values e. Only s75 = 25.0% and C25 = 42.0%; off-pump patients—Me = 18.0%, C75 = 15.0% and C25 = 32.5% (p = 0.022). Additionally, the level of ADP-induced platelet aggregation in patients of this group who received ASA and clopidogrel in the postoperative period on days 8–10 after CABG was significantly lower than in patients who received only ASA: ASA + clopidogrel—Me = 15.5%, C75 = 12.5% and C25 = 21.5%; only ASA—Me = 36.0%, C75 = 27.0% and C25 = 44.0% (p < 0.001). There were no other statistically significant differences in the levels of induced platelet aggregation in sASA and rASA patients with CHD.We investigated the features of platelet aggregation in sASA and rASA patients with CHD depending on the type of surgery (on-pump or off-pump CABG) and postoperative therapy (only ASA or ASA + clopidogrel). It was found that the level of ADP-induced platelet aggregation in sASA patients operated on-pump on the 8–10th day after CABG was higher than in off-pump patients: on-pump CABG—Me = 30.5%, CLevel of ROS synthesis by platelets was determined using two chemiluminescent indicators—lucigenin and luminol. Lucigenin does not penetrate into the cell and interacts only with the superoxide radical, thereby characterizing the activity of the membrane Nox2 ,34. Lumip < 0.05 in all indicators: Tmax, Imax and S). The indicators of lucigenin-enhanced platelet chemiluminescence in rASA patients during the observed period were much less stable. The spontaneous (by indicators Tmax (p = 0.012) and Imax (p = 0.022)) and ADP-induced (by indicators Imax (p = 0.012) and S (p = 0.039)) lucigenin-enhanced platelet chemiluminescence in rASA patients before CABG was lower than in sASA patients. The indicators of lucigenin-dependent chemiluminescence in rASA patients during this period corresponded to the control level. Some indicators of this type of chemiluminescent platelet activity in rASA patients on the first day after CABG were also lower than in sASA patients: Tmax (p < 0.001) and S (p = 0.007) of spontaneous chemiluminescence, Tmax (p = 0.004) and Imax of ADP-induced chemiluminescence (p = 0.029). rASA Patients in this period had an increase in Imax spontaneous chemiluminescence relative to the control level (p = 0.037) as well as a decrease in Tmax relative to initial indicators (p = 0.010). The indices of lucigenin-enhanced platelet chemiluminescence in rASA patients 8–10 days after CABG were also lower than in sASA patients: Tmax (p < 0.001) and Imax (p = 0.021) of spontaneous chemiluminescence, Tmax (p = 0.004) and Imax (p = 0.005) and S (p = 0.014) of ADP-induced chemiluminescence. It was additionally found that the Imax of spontaneous lucigenin-enhanced platelet chemiluminescence in rASA patients during this period was significantly higher than the control (p = 0.042) and initial (p = 0.041) levels.The results of a study of the chemiluminescent activity of platelets in CHD patients are presented in p = 0.026) and induced (p = 0.010) luminol-enhanced chemiluminescence relative to control values was found in sASA patients during this period. A decrease in TmaxT ADP-induced platelet chemiluminescence was also found in rASA patients relative to the control level (p = 0.023). Spontaneous and ADP-induced luminol-enhanced platelet chemiluminescence in sASA and rASA patients on the first day after CABG significantly increased relative to control values (in indicators Imax and S). Tmax was the only indicator of luminol-enhanced platelet chemiluminescence in CHD patients, which varies depending on the sensitivity to ASA during this period of the examination (p = 0.025). Additionally, it was found that Tmax of spontaneous luminol-enhanced platelet chemiluminescence in rASA patients on the first day after CABG increased relative to the initial levels (p = 0.010). Spontaneous and ADP-induced luminol-enhanced chemiluminescence of platelets in sASA patients on the 8–10th day after CABG significantly increased both relative to control values and the initial level (p < 0.05 in Imax and S). In addition, sASA patients during this period of the examination had a longer Tmax of spontaneous (p = 0.015) and induced (p = 0.001) luminol-enhanced platelet chemiluminescence than in people in the control group. Spontaneous luminol-enhanced platelet chemiluminescence in rASA patients 8–10 days after CABG was higher than in control subjects (in Imax p = 0.037 and in S p = 0.020). In addition, rASA patients during this examination period have a higher value of Imax of ADP-induced luminol-enhanced chemiluminescence of platelets compared with the control (p = 0.026) and initial (p = 0.023) levels. Statistically important differences depending on acid resistance were found in CHD patients during this observation period for Tmax on spontaneous (p = 0.036) and ADP-induced (p = 0.021) luminol-enhanced platelet chemiluminescence.Quite other changes in CHD patients were detected by luminol-enhanced platelet chemiluminescence . The indr = −0.27, p = 0.048 and r = −0.28, p = 0.042, accordingly). The level of adrenaline-induced aggregation also negatively correlated with the Imax and S of spontaneous lucigenin-enhanced chemiluminescence of platelets in sASA patients during this observation period . In addition, the level of ARA-induced platelet aggregation with the addition of ASA in sASA patients before CABG was negatively correlated with the Imax of ADP-induced lucigenin-enhanced  and S of spontaneous luminol-enhanced  platelet chemiluminescence. We did not reveal the relationship between the aggregation and chemiluminescent activity of platelets in sASA patients after CABG. Patients with rASA before CABG had positive correlations of ARA-induced aggregation with S of spontaneous  as well as Imax  and S  of ADP-induced luminol-enhanced chemiluminescence of platelets. The level of ADP-induced aggregation in rASA patients on the first day after CABG was also positively correlated with the Imax of spontaneous  and ADP-induced  luminol-enhanced platelet chemiluminescence. The level of ARA-induced aggregation in patients of this group on the 8–10th day after CABG was interrelated with the Imax of spontaneous  and ADP-induced  luminol-enhanced platelet chemiluminescence.The relationships between platelet aggregation and chemiluminescent activity were investigated using correlation analysis. We did not find statistically significant correlation between these indicators in individuals of the control group. It was found that the level of ADP-induced aggregation was negatively correlated with Imax ADP-induced lucigenin- and luminol-enhanced platelet chemiluminescence in sASA patients with CHD before CABG .p < 0.05 in all cases)  and on the first day after CABG  (p < 0.001) and initial (p = 0.008) levels in platelets in sASA patients at 8–10 days after CABG, while the enzyme activity in rASA patients during this observation period corresponded to control values. The activity of the anaerobic reaction of lactate dehydrogenase (NADH-LDH) in platelets is significantly increased in sASA patients with CHD before CABG (p = 0.002) and also on the first (p = 0.002) and 8–10th day (p = 0.004) after CABG relative to control values (p = 0.047). The activity of the NADH-dependent glutamate dehydrogenase reaction (NADH-GluDH) in platelets decreased relative to the initial level on the first (p = 0.042) and 8–10th (p < 0.001) days after CABG only in sASA patients (We investigated the activity of NAD- and NADP-dependent dehydrogenases in sASA and rASA platelets with CHD in patients before and after CABG. It was found that the activity of glucose-6-phosphate dehydrogenase (Glu6PDH) in the platelets of CHD patients before and after CABG was significantly lower than in individuals in the control group (l cases) a. The acrdingly) b. The acl values c. The acpatients d. No staThe activity of enzymes in platelets in sASA and rASA patients with CHD did not differ significantly depending on the use of on-pump CABG and the type of postoperative treatment (only ASA or ASA + clopidogrel).r = 0.33, p = 0.014), while this type of aggregation was negatively correlated with NADP-dependent isocitrate dehydrogenase (NADP-ICDH) activity in rASA patients  during this examination period. The level of ARA-induced platelet aggregation in sASA patients on the first day after CABG was positively correlated with the activity of the NADH-GluDH , while the level of ARA-induced aggregation with the addition of ASA already negatively correlated with the activity of the glucose-6-phosphate dehydrogenase (Glu6PDH) in these patients . Glutathione reductase (GR) activity was positively correlated with the levels of ADP- and ARA-induced platelet aggregation  in rASA patients on the 1st day after CABG, while NADH-GluDH activity was already negatively correlated with these types of platelet aggregation activity . The NADH-LDH activity was positively correlated with the level of ARA-induced platelet aggregation in sASA patients  on the 8–10th day after CABG, and this enzyme was negatively correlated with the level of ADP-induced platelet aggregation in rASA patients  during this observation period.The relationships between platelet aggregation ability and enzyme activity were investigated using correlation analysis. People in the control group did not have statistically significant correlations between these indicators. The level of ADP-induced platelet aggregation was positively correlated with NADPH-GluDH activity in sASA patients before CABG . In general, platelet activation is significantly dependent on the intensity of the substrate flow through glycolysis . MoreoveIt should be noted that understanding the metabolic mechanisms of ASA resistance is also important for several other diseases, in particular type 2 diabetes mellitus (T2DM). It is known that T2DM is accompanied by an increased risk of thrombosis due to increased functional activity of platelets ,48,49. IThus, the aggregation activity of platelets depended on the sensitivity of CHD patients to ASA and decreased during postoperative ASA therapy. The activity of ARA-induced platelet aggregation most differentially differentiated CHD patients depending on their sensitivity to ASA. Platelet aggregation activity induced by ADP, collagen and adrenaline did not differ in patients, but decreased during postoperative ASA treatment. The state of platelet metabolism in sASA and rASA patients with CHD varied both before and after CABG. The most pronounced differences in metabolic parameters of platelets in sASA and rASA patients were detected by Nox2 activity. rASA patients had low oxidase activity compared with sASA patients. The synthesis of secondary ROS by platelets of CHD patients did not depend on the sensitivity of patients to ASA but increased during postoperative treatment with ASA. The activity of NAD(P)-dependent dehydrogenases in platelets did not differ in sASA and rASA patients with CHD. However, the activity of dehydrogenases in platelets in sASA patients was more sensitive to postoperative ASA therapy. Moreover, dehydrogenases involved in energy processes were more sensitive to the effects of ASA. Due to the fact that metabolic intermediates (ROS and other products of enzymatic reactions) not only have a metabolic effect but also affect regulatory and epigenetic processes, we can conclude that sensitivity to ASA can be determined by platelet metabolism.Sixty-six patients with stable angina pectoris, grade II–III according to the Canadian Cardiovascular Society classification were enrolled in the study (49 men and 17 women) . The conp < 0.001.The study protocol was approved by the Ethics Committee of the Krasnoyarsk State Medical University (protocol №76/2016 from 04/05/2017). It was conducted in accordance with the Declaration of Helsinki and was consistent with applicable guidelines for good clinical practice. Written consent was obtained from all participants. During the hospitalization, all patients received therapy according to the Russian Cardiology Society guidelines. The antiplatelet therapy was stopped for at least 5 days before CABG. In the post-surgical period, enrolled patients were treated with 100 mg of enteric form ASA along (39 patients) either 100 mg ASA + clopidogrel 75 mg per day (27 patients). In all patients ASA was started on the first day after CABG. Patients took following medications before CABG: β-blockers—92.4% of patients, Angiotensin-converting enzyme inhibitors—90.9%, Angiotensinogen II receptor blockers—5%, Calcium channel blockers—16.9%, Diuretics—42.4%, Aldosterone antagonists—40.6%, Statins—100%. Median of heparin dose in the group of patients during on-pump CABG 240 mg (220–300), during off-pump CABG—370 mg (320–445), Forty-three (65.2%) patients had stable angina grade II, 23 (34.8%) patients had grade III of angina pectoris. The age was from 36 to 72 years (mean age 60.9 ± 6.9 years). All patients had atherosclerotic lesions of coronary arteries, proven by coronaroangiography. The baseline characteristic is shown in The venous blood samples were acquired from patients before coronary artery bypass grafting (CABG) after five days of ASA withdrawal, on the first day after surgery and on the 8–10th day after surgery. We have selected these time points to estimate platelet function and metabolism in stable condition before CABG, on the first day after on-pump or off-pump CABG to realize its influence on platelets and on the 8–10th day after surgery as the last day of treatment in a hospital before discharge.Twelve patients (18.2%) were resistant to ASA.9 platelets/L with buffer . Evaluation of the number of the isolated platelets was carried out on a hematological analyzer Sysmex XE-5000  by a standard method [Blood samples were collected from the antecubital vein in vacutainer tube with 3.8% tri-sodium citrate, in the morning and on an empty stomach. Blood was centrifuged for 10 min at 140 g at room temperature to produce platelet-rich plasma. The supernatant was carefully transferred into a plastic tube and adjusted to 300 × 10d method . Platelev/v) was centrifuged for 10 min at 140× g at room temperature to produce the platelet-rich plasma. The platelet-poor plasma was obtained by further centrifuging the remainder of the platelet-rich plasma specimen at 700× g for 20 min at room temperature. Platelet aggregation was determined on an optical aggregometer 490 Chrono-Log (USA) with a self-calibration system. 500 μL platelet-poor plasma was placed in an aggregometer comparison cuvette. Next, 500 μL of platelet-rich plasma was added to a cuvette of an aggregometer with a magnetic stirrer. Samples were incubated for 2 min at a temperature of 37 °C. Then, the aggregation inducer was added to the platelet-rich plasma cuvette and the level of aggregation was measured relative to the cuvette with platelet-poor plasma for 5–6 min. Collagen (2 μg/mL), ADP (5 μM), adrenaline (10 μM) and ARA (0.5 mM) were used as platelet aggregation inducers. Platelet aggregation was expressed as a percentage.Blood with 3.8% tri-sodium citrate . The resulting cell suspension was centrifuged for 15 min at 400× g. The cell pellet was resuspended in 10 mL of buffer A and centrifuged again for 1 min at 400× g. Then, 9 mL of the supernatant was collected and centrifuged again for 15 min at 400× g. The supernatant was carefully removed and 10 mL of buffer B  was added to the pellet. The cell suspension was centrifuged in the same mode. The supernatant was removed and the pellet was diluted in 400 μL of buffer B and centrifuged for 1 min at 140× g. Platelet-containing supernatant was transferred to a plastic tube and used for further investigations. Evaluation of the number and purity of the isolated platelets was carried out on a hematological analyzer Sysmex XE-5000  by a standard method [Platelet-rich plasma was obtained by centrifuging stabilized blood (sodium citrate) at 140× d method . The pur8 platelets per sample were used to conduct a chemiluminescent reaction. The composition of the reaction sample to determine spontaneous platelet chemiluminescence also included 50 μL of lucigenin (50 μg/mL) or luminol (50 μg/mL) and 250 μL of buffer B . The sample composition for determining ADP-induced platelet chemiluminescence included platelets and chemiluminescent indicators in the same amount, 200 μL of buffer B and 50 μL of 0.1 M ADP. Evaluation of spontaneous and ADP-induced platelet chemiluminescence was performed for 90 min on the 36-channel biochemiluminescence analyzer BLM-3607 . The following indicators of the chemiluminescent platelet reaction were determined: time to maximum (Tmax), maximum intensity (Imax) and the area (S) under the chemiluminescence curve. The enhancement of the chemiluminescence of induced ADP (activation index—AI) was determined by the ratio of the area under the curve of ADP-induced chemiluminescence to the area under the curve of spontaneous chemiluminescence.10−glucose-6-phosphate dehydrogenase (Glu6PDH);−glycerol-3-phosphate dehydrogenase (Gly3PDH);−NADP-dependent malate dehydrogenase decarboxylated (NADP-MDH);−lactate dehydrogenase—according to NAD- and NADH-dependent reactions ;−malate dehydrogenase—according to NAD- and NADH-dependent reactions ;−NADP-dependent glutamate dehydrogenase—according to NADP- and NADPH-dependent reactions ;−NAD-dependent glutamate dehydrogenase—according to NAD- and NADH-dependent reactions ;−NADP-dependent isocitrate dehydrogenase (NADP-ICDH);−NAD-dependent isocitrate dehydrogenase (NAD-ICDH);−glutathione reductase (GR).The activity of NAD- and NADP-dependent dehydrogenases in platelets was determined using the bioluminescent method . The actPhotobacterium leiognathi  [The platelets were previously destroyed by osmotic lysis (1:5 by volume) with addition of 2 mM dithiotreitol . Bioluminescent analysis was performed using NAD(P):FMN oxidoreductase-luciferase  from noyarsk) . Activitnoyarsk) . Protein25—C75). Significance of differences between indicators was assessed by nonparametric criterion Mann-Whitney U test. The statistical significance of the differences in the dynamics of treatment (dependent variables) was determined by the Wilcoxon matched pairs test. To compare categorical variables, the χ2 test was used, except when expected frequencies in contingency tables were less than 5, in which case the Fisher exact test was used. Spearman rank correlation coefficients were calculated to characterize the strength of the relationship between platelet aggregation activity and metabolic parameters. Statistical analysis was performed in an application package Statistica 6.1 .Statistical description was performed by counting the median (Me) and the interquartile ranges in the form of 25 and 75 percentiles (C